Substrate targeting mechanism of a DesCEND pathway

摘要

Abstract This request for a supplement to the grant R01GM135592 proposes the acquisition of a liquid chromatography protein purification system to support the research aims that focus on investigating the substrate targeting mechanisms of the F-box subunit FBXO31 of the SCF ubiquitin ligase in a novel DesCEND pathway. By identifying and characterizing substrates of FBXO31 at both a systems level and the molecular level, we proposed to elucidate the complementary structural determinants of the FBXO31–degron pair that confer substrate specificity and dictate protein recognition and fate. To achieve our goals, it is vital that we have the ability to obtain highly purified protein samples from complex starting materials derived from cell lysates and recombinant protein expression systems. This proposal requests funds to replace a 14-year old (and recently defunct) AKTA FPLC protein purification instrument with a similar but up-to-date version from the same manufacturer. The proposed system, the AKTA pure, is a versatile and powerful liquid chromatography system for the reliable purification of proteins and protein complexes. It covers a broad range of chromatography applications and meets the automation requirements needed to deliver the highest purity. The instrument will ensure the continued biochemical, biophysical and structural studies proposed in the parent award.